Noradrenergic Nerve Research Articles

A pathophysiological biomarker combination separates Lewy body from non-Lewy body neurogenic orthostatic hypotension ​.

Neurogenic orthostatic hypotension (nOH) results from deficient reflexive delivery of norepinephrine to cardiovascular receptors in response to decreased cardiac venous return. Lewy body (LB) forms of nOH are characterized by low 18F-dopamine-derived radioactivity (a measure of cardiac noradrenergic deficiency), olfactory dysfunction by the University of Pennsylvania Smell Identification Test (UPSIT), and increased deposition of alpha-synuclein (α-syn) in dermal sympathetic noradrenergic nerves by the α-syn-tyrosine hydroxylase (TH) colocalization index. This observational, cross-sectional study explored whether combinations of these biomarkers specifically identify LB forms of nOH. Clinical laboratory data were reviewed from patients referred for evaluation at the National Institutes of Health for chronic autonomic failure between 2011 and 2023. The cutoff value for low myocardial 18F-dopamine-derived radioactivity was 6000 nCi-kg/cc-mCi, for olfactory dysfunction an UPSIT score ≤ 28, and for an increased α-syn-TH colocalization index ≥ 1.57. A total of 44 patients (31 LB, 13 non-LB nOH) had data for all three biomarkers. Compared to the non-LB group, the LB nOH group had low myocardial 18F-dopamine-derived radioactivity, low UPSIT scores, and high α-syn-TH colocalization indexes (p < 0.0001 each). Combining the three biomarkers completely separated the groups. Cluster analysis identified two distinct groups (p < 0.0001) independently of the clinical diagnosis, with one cluster corresponding exactly to LB nOH. LB forms of nOH feature cardiac noradrenergic deficiency, olfactory dysfunction, and increased α-syn-TH colocalization in skin biopsies. Combining the data for these variables efficiently separates LB from non-LB nOH. Independently of the clinical diagnosis, this biomarker triad identifies a pathophysiologically distinct cluster of nOH patients.

NADPH oxidase 2 mediates cardiac sympathetic denervation and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced cardiomyopathy

Doxorubicin has been used extensively as a potent anticancer agent, but its clinical use is limited by its cardiotoxicity. However, the underlying mechanisms remain to be fully elucidated. In this study, we tested whether NADPH oxidase 2 (Nox2) mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced heart failure. Nox2 knockout (KO) and wild-type (WT) mice were randomly assigned to receive a single injection of doxorubicin (15 mg/kg, i.p.) or saline. WT doxorubicin mice exhibited the decreases in survival rate, left ventricular (LV) wall thickness and LV fractional shortening and the increase in the lung wet-to-dry weight ratio 1 week after the injections. These alterations were attenuated in Nox2 KO doxorubicin mice. In WT doxorubicin mice, myocardial oxidative stress was increased, myocardial noradrenergic nerve fibers were reduced, myocardial expression of PGP9.5, GAP43, tyrosine hydroxylase and norepinephrine transporter was decreased, and these changes were prevented in Nox2 KO doxorubicin mice. Myocyte autophagy was increased and myocyte size was decreased in WT doxorubicin mice, but not in Nox2 KO doxorubicin mice. Nox2 mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy—both of which contribute to cardiac atrophy and failure after doxorubicin treatment.

Hepatic Innervations and Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. Increased sympathetic (noradrenergic) nerve tone has a complex role in the etiopathomechanism of NAFLD, affecting the development/progression of steatosis, inflammation, fibrosis, and liver hemodynamical alterations. Also, lipid sensing by vagal afferent fibers is an important player in the development of hepatic steatosis. Moreover, disorganization and progressive degeneration of liver sympathetic nerves were recently described in human and experimental NAFLD. These structural alterations likely come along with impaired liver sympathetic nerve functionality and lack of adequate hepatic noradrenergic signaling. Here, we first overview the anatomy and physiology of liver nerves. Then, we discuss the nerve impairments in NAFLD and their pathophysiological consequences in hepatic metabolism, inflammation, fibrosis, and hemodynamics. We conclude that further studies considering the spatial-temporal dynamics of structural and functional changes in the hepatic nervous system may lead to more targeted pharmacotherapeutic advances in NAFLD.

TRPV1 is involved in abdominal hyperalgesia in a mouse model of lipopolysaccharide-induced peritonitis and influences the immune response via peripheral noradrenergic neurons

AimsThe transient receptor potential vanilloid subfamily 1 (TRPV1) not only plays a role as a nociceptor but also has some regulatory effects on the immune system. We investigated the effects of TRPV1 on abdominal pain and the immune system in lipopolysaccharide (LPS)-induced peritonitis and the association between TRPV1 and peripheral noradrenergic neurons. Main methodsExperiments were performed in 8- to 14-week-old male wild-type (WT) and TRPV1 knockout (KO) mice. The mice were intraperitoneally injected with a non-lethal dose of LPS. Pain assessment and investigation of changes in the immune system were performed. Denervation of sympathetic nerves and the noradrenergic splenic nerve was induced by intraperitoneal administration of 6-hydroxydopamine. Key findingsThe levels of serum cytokines were not significantly different in WT mice and TRPV1 KO mice. Abdominal mechanical hyperalgesia was greater in WT mice than in TRPV1 KO mice from 6 h to 3 days. The numbers of macrophages, neutrophils, dendritic cells, and CD4 T cells in the spleens of TRPV1 KO mice were significantly increased compared to those in WT mice 4 days after LPS administration. By noradrenergic denervation, the numbers of those cells in WT mice increased to levels comparable to those in TRPV1 KO mice. SignificanceIn LPS-induced peritonitis, abdominal inflammatory pain was transmitted via TRPV1. In addition, TRPV1 had an anti-inflammatory effect on the spleen in the late phase of peritonitis. This anti-inflammatory effect was thought to be mediated by activation of the sympathetic nervous system and/or noradrenergic splenic nerve induced by TRPV1 activation.

Sympathetic innervation of human and porcine spleens: implications for between species variation in function

BackgroundThe vagus nerve affects innate immune responses by activating spleen-projecting sympathetic neurons, which modulate leukocyte function. Recent basic and clinical research investigating vagus nerve stimulation to engage the cholinergic anti-inflammatory pathway (CAP) has shown promising therapeutic results for a variety of inflammatory diseases. Abundant sympathetic innervation occurs in rodent spleens, and use of these species has dominated mechanistic research investigating the CAP. However, previous neuroanatomical studies of human spleen found a more restricted pattern of innervation compared to rodents. Therefore, our primary goal was to establish the full extent of sympathetic innervation of human spleens using donor tissue with the shortest procurement to fixation time. Parallel studies of porcine spleen, a large animal model, were performed as a positive control and for comparison.MethodsHuman and porcine spleen tissue were fixed immediately after harvest and prepared for immunohistochemistry. Human heart and porcine spleen were stained in conjunction as positive controls. Several immunohistochemical protocols were compared for best results. Tissue was stained for tyrosine hydroxylase (TH), a noradrenergic marker, using VIP purple chromogen. Consecutive tissue slices were stained for neuropeptide Y (NPY), which often co-localizes with TH, or double-labelled for TH and CD3, a T cell marker. High-magnification images and full scans of the tissue were obtained and analyzed for qualitative differences between species.ResultsTH had dominant perivascular localization in human spleen, with negligible innervation of parenchyma, but such nerves were abundant throughout ventricular myocardium. In marked contrast, noradrenergic innervation was abundant in all regions of porcine spleen, with red pulp having more nerves than white pulp. NPY stain results were consistent with this pattern. In human spleen, noradrenergic nerves only ran close to T cells at the boundary of the periarterial lymphatic sheath and arteries. In porcine spleen, noradrenergic nerves were closely associated with T cells in both white and red pulp as well as other leukocytes in red pulp.ConclusionSympathetic innervation of the spleen varies between species in both distribution and abundance, with humans and pigs being at opposite extremes. This has important implications for sympathetic regulation of neuroimmune interactions in the spleen of different species and focused targeting of the CAP in humans.

Modeling the Progression of Cardiac Catecholamine Deficiency in Lewy Body Diseases.

BackgroundLewy body diseases (LBDs) feature deficiency of the sympathetic neurotransmitter norepinephrine in the left ventricular myocardium and sympathetic intra‐neuronal deposition of the protein alpha‐synuclein (αS). LBDs therefore are autonomic synucleinopathies. Computational modeling has revealed multiple functional abnormalities in residual myocardial sympathetic noradrenergic nerves in LBDs, including decreased norepinephrine synthesis, vesicular storage, and recycling. We report an extended model that enables predictions about the progression of LBDs and effects of genetic predispositions and treatments on that progression.Methods and ResultsThe model combines cardiac sympathetic activation with autotoxicity mediated by the dopamine metabolite 3,4‐dihydroxyphenylacetaldehyde. We tested the model by its ability to predict longitudinal empirical data based on cardiac sympathetic neuroimaging, effects of genetic variations related to particular intra‐neuronal reactions, treatment by monoamine oxidase inhibition to decrease 3,4‐dihydroxyphenylacetaldehyde production, and post‐mortem myocardial tissue contents of catecholamines and αS. The new model generated a triphasic decline in myocardial norepinephrine content. This pattern was confirmed by empirical data from serial cardiac 18F‐dopamine positron emission tomographic scanning in patients with LBDs. The model also correctly predicted empirical data about effects of genetic variants and monoamine oxidase inhibition and about myocardial levels of catecholamines and αS.ConclusionsThe present computational model predicts a triphasic decline in myocardial norepinephrine content as LBDs progress. According to the model, disease‐modifying interventions begun at the transition from the first to the second phase delay the onset of symptomatic disease. Computational modeling coupled with biomarkers of preclinical autonomic synucleinopathy may enable early detection and more effective treatment of LBDs.

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

SummaryThe autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects.

Localization of NGF expression in mouse spleen and salivary gland: Relevance to pleotropic functions

Our primary goal was to determine if leukocytes are a source of nerve growth factor (NGF) in mouse spleen. Noradrenergic nerves were localized to arteries and white pulp in normal spleens but only to arteries in ultra-immunodeficient R2G2 mice that lack leukocytes. NGF mRNA was detected in vascular cells and leukocytes of normal spleen, and several of the latter were T cells based on double labeling for NGF mRNA and CD3. Our findings indicate NGF is produced by vascular cells and to a lesser extent by leukocytes in spleen and provide support for pleiotropic actions in spleen and salivary glands.

Pathophysiological significance of increased \u03b1-synuclein deposition in sympathetic nerves in Parkinson\u2019s disease: a post-mortem observational study

BackgroundParkinson’s disease (PD) is characterized by intra-neuronal deposition of the protein α-synuclein (α-syn) and by deficiencies of the catecholamines dopamine and norepinephrine (NE) in the brain and heart. Accumulation of α-syn in sympathetic noradrenergic nerves may provide a useful PD biomarker; however, whether α-syn buildup is pathophysiological has been unclear. If it were, one would expect associations of intra-neuronal α-syn deposition with catecholaminergic denervation and with decreased NE contents in the same samples.MethodsWe assayed immunoreactive α-syn and tyrosine hydroxylase (TH, a marker of catecholaminergic innervation) concurrently with catecholamines in coded post-mortem scalp skin, submandibular gland (SMG), and apical left ventricular myocardial tissue samples from 14 patients with autopsy-proven PD and 12 age-matched control subjects who did not have a neurodegenerative disease.ResultsThe PD group had increased α-syn in sympathetic noradrenergically innervated arrector pili muscles (5.7 times control, P < 0.0001), SMG (35 times control, P = 0.0011), and myocardium (11 times control, P = 0.0011). Myocardial TH in the PD group was decreased by 65% compared to the control group (P = 0.0008), whereas the groups did not differ in TH in either arrector pili muscles or SMG. Similarly, myocardial NE was decreased by 92% in the PD group (P < 0.0001), but the groups did not differ in NE in either scalp skin or SMG.ConclusionsPD entails increased α-syn in skin, SMG, and myocardial tissues. In skin and SMG, augmented α-syn deposition in sympathetic nerves does not seem to be pathogenic. The pathophysiological significance of intra-neuronal α-syn deposition appears to be organ-selective and prominent in the heart.

Brown Fat Anatomy in Humans and Rodents.

The Brown Adipose Tissue (BAT) is composed by mitochondrial rich, multilocular adipocytes, in strict topographical and functional relation with vasculature and noradrenergic nerves. Brown adipocytes are able to dissipate energy to produce heat, in a process known as non-shivering thermogenesis. Due to its contribution to energy expenditure, BAT is intensely studied for its potential to counteract metabolic diseases such as obesity, type 2 diabetes, dyslipidemia and cardiovascular diseases. BAT displays specific morphological characteristics that allow to assess its functional state. In this chapter we describe methodologies to properly dissect BAT depots, evaluate their gross anatomy, and assess its activation by light microscopy using peroxidase immunostaining and by laser scanning confocal microscopy using immunofluorescence. We also describe methodologies to study BAT ultrastructure by transmission and scanning electron microscopy, to visualize peroxidase immunostaining reactions at an ultrastructural level and to perform immunofluorescence reactions on paraffin-embedded samples, more often available in the clinical setting (due to the possibility to store them long-term) as opposed to fresh samples. The described techniques can be employed to study BAT morphology and activation in response to various stimuli (e.g., cold exposure; specific dietary composition) and in different pathological conditions (e.g., obesity; type 2 diabetes).

Hormonal and Neurological Aspects of Dog Walking for Dog Owners and Pet Dogs.

The hormone oxytocin is involved in various aspects of the relationship between humans and animals. Dog walking is a common activity for dog owners and their dogs. The walk, of course, should be good for the health of the dog as well as its owner. In Experiment I, we assessed whether salivary oxytocin and cortisol in dog owners changed because of walking their dogs. Ten owners walked with their dogs and walked alone. Similar to other previous research, walking with a dog did not significantly change oxytocin and cortisol. Therefore, in Experiment II, we investigated the effect of dog walking on brain noradrenergic and GABAergic neural activity, as indicated by salivary MHPG and GABA, in 14 dog owners. Walking with a dog reduced salivary MHPG compared to walking alone, and MHPG was correlated negatively with GABA. Thus, dog walking activated GABAergic nerves in the brain and suppressed noradrenergic nerves, effectively relieving stress.

α-Synuclein Deposition in Sympathetic Nerve Fibers in Genetic Forms of Parkinson's Disease.

Cytoplasmic inclusions of α-synuclein (α-syn) in brainstem neurons are characteristic of idiopathic Parkinson's disease (PD). PD also entails α-syn buildup in sympathetic nerves. Among genetic forms of PD, the relative extents of sympathetic intraneuronal accumulation of α-syn have not been reported. This cross-sectional observational study compared magnitudes of intraneuronal deposition of α-syn in common and rare genetic forms of PD. α-Syn deposition was quantified by the α-syn-tyrosine hydroxylase colocalization index in C2 cervical skin biopsies from 65 subjects. These included 30 subjects with pathogenic mutations in SNCA (n=3), PRKN [biallelic (n=7) and monoallelic (n=3)], LRRK2 (n=7), GBA (n=7), or PARK7/DJ1 [biallelic (n=1) and monoallelic (n=2)]. Twenty-five of the mutation carriers had PD and five did not. Data were also analyzed from 19 patients with idiopathic PD and 16 control participants. α-Syn deposition varied as a function of genotype (F=16.7, P< 0.0001). It was above the control range in 100% of subjects with SNCA mutations, 100% with LRRK2 mutations, 95% with idiopathic PD, 83% with GBA mutations, and 0% with biallelic PRKN mutations. α-Syn deposition in the biallelic PRKN group was significantly higher than in the control group. In addition, patients with biallelic PRKN mutations had higher α-syn deposition than their unaffected siblings. Individuals with SNCA, DJ-1, LRRK2, or GBA mutations have substantial intraneuronal α-syn deposition in sympathetic noradrenergic nerves in skin biopsies, whereas those with biallelic PRKN mutations do not. Biallelic PRKN patients may have mildly increased α-syn deposition compared with control subjects. © 2021 International Parkinson and Movement Disorder Society.

Subsecond spontaneous catecholamine release in mesenteric lymph node ex vivo.

Measuring the dynamics of neurochemical-regulated immunity, particularly in the gut, has been a growing interest over the last several years because of its important implications in gastrointestinal inflammation, neurodegeneration, and even depression. Sympathetic noradrenergic nerves innervate the gastrointestinal tract and resident immune organs, including the mesenteric lymph nodes (MLN) and Peyer's patches. Previous research has suggested that neuronal inputs in the MLN release norepinephrine (NE) at neural-immune synapses to regulate immune function. The current immunological techniques do not have the appropriate temporal or spatial resolution to monitor this dynamic process in real-time, within specific regions of intact lymphoid organs. Monitoring dynamic neural signaling within intact immune organs, in real-time, would facilitate a deeper understanding of neuroimmune communication and would allow the mechanism of rapid immunomodulation to be elucidated. Here, we overcome this technological barrier by coupling real-time neurochemical detection using fast-scan cyclic voltammetry (FSCV) in live MLN slices from C57BL/6 mice. We have discovered rapid, spontaneous catecholamine transients in the T-cell zone of the MLN which are on the order of a few hundred nanomolar, rapid (a few seconds), and frequent (every 20-s). We demonstrate that the β2 -adrenergic receptor and the classic catecholamine transporters (DAT and NET) play a minor role in transient regulation in the MLN suggesting that regulation at the neural-immune synapse is quite complicated and further mechanistic studies are needed. Overall, these findings provide direct evidence for rapid neurochemical events in the MLN which could have a major impact on our understanding of neurochemical-regulated immunomodulation in the gut.

The Release of Norepinephrine in C57BL/6J Mice Treated with 6-Hydroxydopamine (6-OHDA) is Associated with Translocations in Enteric Escherichia coli via the QseC Histidine Kinase Receptor.

BackgroundWe aimed to investigate the effects of norepinephrine (NE) released from endogenous stores on bacterial translocation of Escherichia coli in mice by administration of 6-hydroxydopamine (6-OHDA), which selectively destroys noradrenergic nerve terminals.Material/MethodsE. coli strain BW25113 and its derivatives (BW25113ΔqseC and BW25113ΔqseC pQseC) were used in this study. The serum concentrations of endotoxin were analyzed. The strains BW25113, BW25113ΔqseC, and BW25113ΔqseC pQseC were detected respectively in tissue specimens harvested from mice treated with 6-OHDA.ResultsMice treated with BW25113ΔqseC showed reduced levels of bacterial translocation following administration of 6-OHDA compared with mice treated with BW25113. The defect of E. coli QseC receptor caused the norepinephrine-QseC signal chain to be interrupted, and the invasiveness and penetrating power of the bacteria on the intestinal mucosa was weakened, eventually leading to a significant decrease in the incidence of bacterial translocation.ConclusionsNE modulates the interaction of enteric bacterial pathogens with their hosts via QseC. The blockade of the QseC receptor-mediated effects may be useful to attenuate bacterial translocation.

Decreased neural expression of the noradrenaline transporter in the papillary dermis after partial sciatic nerve lesion

After peripheral nerve injury, regeneration or collateral sprouting of noradrenergic nerve fibres in the papillary dermis of the injured limb may contribute to sympathetically-maintained pain. The aim of this study was to determine whether noradrenergic nerve fibre regeneration after partial sciatic nerve ligation (PSL) in Wistar rats was accompanied by parallel shifts in expression of the noradrenaline transporter (NAT). Four or 28 days after PSL surgery, immunohistochemistry was used to examine NAT expression in plantar hind paw skin in relation to pan-neuronal markers (class III beta-tubulin and protein gene product 9.5), peptidergic afferents containing calcitonin gene-related peptide (CGRP), nonpeptidergic afferents labelled by isolectin B4 (IB4), and tyrosine hydroxylase (TH), a marker for cutaneous noradrenergic nerve fibres. Most dermal nerve fibre populations decreased shortly after PSL. However, four weeks after PSL, an increase in staining intensity of CGRP and novel expression of TH were observed in the papillary dermis on the injured side. In contrast, neural expression of NAT was reduced in this region. Loss of NAT might have implications for sympathetically-maintained pain, as failure to rapidly clear noradrenaline could exacerbate aberrant sympathetic-sensory signalling between closely apposed noradrenergic and peptidergic nerve fibres.

Brain control of humoral immune responses amenable to behavioural modulation

It has been speculated that brain activities might directly control adaptive immune responses in lymphoid organs, although there is little evidence for this. Here we show that splenic denervation in mice specifically compromises the formation of plasma cells during a T cell-dependent but not T cell-independent immune response. Splenic nerve activity enhances plasma cell production in a manner that requires B-cell responsiveness to acetylcholine mediated by theα9 nicotinic receptor, and T cells that express choline acetyl transferase1,2 probably act as a relay between the noradrenergic nerve and acetylcholine-responding B cells. We show that neurons in the central nucleus of the amygdala (CeA) and the paraventricular nucleus (PVN) that express corticotropin-releasing hormone (CRH) are connected to the splenic nerve; ablation or pharmacogenetic inhibition of these neurons reduces plasma cell formation, whereas pharmacogenetic activation of these neurons increases plasma cell abundance after immunization. In a newly developed behaviour regimen, mice are made to stand on an elevated platform, leading to activation of CeA and PVN CRH neurons and increased plasma cell formation. In immunized mice, the elevated platform regimen induces an increase in antigen-specific IgG antibodies in a manner that depends on CRH neurons in the CeA and PVN, an intact splenic nerve, and B cell expression of the α9 acetylcholine receptor. By identifying a specific brain-spleen neural connection that autonomically enhances humoral responses and demonstrating immune stimulation by a bodily behaviour, our study reveals brain control of adaptive immunity and suggests the possibility to enhance immunocompetency by behavioural intervention.

SPARC: Autonomic Innervation of Porcine Ventricular Myocardium and Purkinje Fibers

Previous studies demonstrated that porcine ventricular myocardium receives abundant innervation, but most of this work focused on nerves at epicardial and endocardial surfaces. Furthermore, our knowledge regarding cholinergic innervation of ventricular muscle remains sparse. The goal of this study was to identify and quantify the regional abundance of cholinergic and noradrenergic nerves in left and right ventricular walls from the base of the heart to the apex. One‐year old male and female Yucatan minipigs were sacrificed, hearts were removed, and blood was flushed from the tissue by infusing heparinized saline into the coronary arteries. Samples of myocardium were collected from regions of interest, and these were immersion‐fixed overnight in cold 4% paraformaldehyde/0.2% picric acid in buffer before cryoprotection. Transmural 30 μm sections were cut in a cryostat and collected on slides for labeling of nerve fibers by fluorescence immunohistochemistry. Adjacent sections were stained for the pan‐neuronal marker protein gene product 9.5 (PGP9.5) or double‐labeled for vesicular acetylcholine transporter (VAChT) and tyrosine hydroxylase (TH) to identify cholinergic and noradrenergic nerves, respectively. Digital images of labeled sections were collected and deconvoluted using an Olympus BX41 microscope with a 20X objective, DP74 camera, and cellSense Dimension software. Nerve densities were determined as % area using ImageJ software. Abundant labeling for all markers was detected across the ventricular walls, and nerve densities were comparable at all levels from base to apex. Total nerve density based on PGP9.5 staining ranged from ~2.5 to 4 % area in the left ventricles, and the sum of TH and VAChT nerve densities accounted for most of the total. For the majority of sections on the left, TH nerves were somewhat more abundant than VAChT nerves, but average cholinergic nerve density was at least 1% area. The ratio of noradrenergic/cholinergic nerves was ~1.5 to 2. Preliminary evidence indicates cholinergic and noradrenergic nerves have similar densities in right ventricle. Staining of sections for the glial cell marker S100 showed that Schwann cells were present throughout the ventricles. Purkinje fibers were identified at the endocardium of many sections, and these cells stained intensely for PGP9.5 but did not stain for another neuronal marker microtubule associated protein 2. The Purkinje cells received abundant cholinergic innervation with single nerves occasionally penetrating fiber bundles and wrapping around Purkinje cells. In contrast, noradrenergic innervation of Purkinje fibers was sparse. Our findings provide definitive evidence for prominent and ubiquitous innervation of porcine ventricular muscle by both noradrenergic and cholinergic nerves, providing a basis for balanced regulation of contractile function. In marked contrast, cholinergic innervation is more abundant than noradrenergic in the Purkinje system, suggesting a dominant role of parasympathetic nerves in controlling impulse propagation via the cardiac conducting system.Support or Funding InformationSupported by NIH 3OT2OD023848

Zamicastat is a noncompetitive Dopamine‐β‐hydroxylase inhibitor that modulates sympathetic nervous system activity

Dopamine‐β‐hydroxylase (DβH; EC 1.14.17.1) is an enzyme responsible for the conversion of dopamine (DA) into norepinephrine (NE). It is present in the chromaffin cells of adrenal medulla and in noradrenergic nerve terminals of the central and peripheral sympathetic nervous system. DβH inhibition has been proposed as an alternative mechanism for the treatment of hypertension and chronic heart failure. Zamicastat ((R)‐5‐(2‐(benzylamino)ethyl)‐1‐(6,8‐difluorochroman‐3‐yl)‐1H‐imidazole‐2(3H)‐thione) is a novel DβH inhibitor. The aim of this study was to characterize the pharmacodynamics of Zamicastat in vivo, both in Wistar Han and in spontaneously hypertensive rats (SHR), and on DβH in human cells. DβH activity was measured by a modification of the Nagatsu and Udenfriend (1) spectrophotometric method, based on the conversion of tyramine into octopamine either in adrenal tissues of rats administered orally with Zamicastat (30 mg/Kg) or vehicle, or in human neuroblastoma (SK‐N‐SH) cells. Zamicastat pharmacodynamic characterization in vivo was performed in Wistar rats by evaluating NE and DA levels in tissues using HPLC with electrochemical detection at 9h post‐administration. Additionally, systolic and diastolic blood pressure, heart rate and activity were measured continually by telemetry in SHR rats (a model of essential hypertension) and their controls, Wistar Kyoto (WKY). Zamicastat was found to be a reversible noncompetitive inhibitor of DβH towards tyramine, with a Ki value of 43 (21; 66) nM and either noncompetitive or uncompetitive towards ascorbic acid. Zamicastat (30 mg/Kg, p.o.) fully inhibits adrenal DβH activity from 2 to 8h recovering to baseline values after 24h. This is accompanied with a decrease in NE and an increase in DA in peripheral tissues, namely left ventricle and kidney but not significantly in the central nervous system (brain stem or prefrontal‐cortex). Zamicastat at 3, 30 and 100 mg/Kg displayed a dose‐dependent decrease on systolic and diastolic blood pressure with no effect in heart rate or overall activity when evaluated in SHR rats by telemetry. In conclusion, Zamicastat is a DβH inhibitor displaying binding affinity for both the free enzyme and the complex enzyme‐substrate, that when orally administered at 30 mg/Kg to rats acts peripherally and significantly reduces sympathetic activity by decreasing NE levels and consequently systolic and diastolic blood pressure in an animal model of essential hypertension.

Isolation and Molecular Identification of a Novel Tyramine-producing Bacterium, &amp;lt;i&amp;gt;Rummeliibacillus pycnus&amp;lt;/i&amp;gt;

Decarboxylation of tyrosine through tyrosine decarboxylase enzyme produces tyramine which may represent a serious threat to public health, as it may cause severe toxicological effects. Intake of tyramine will invariably result in acute symptoms as it can rapidly gain access to the bloodstream and to various organs where it can act as a vasoactive agent through interaction with the sympathetic noradrenergic nerve terminals innervating cardiac and vascular smooth muscle tissues. This research is focusing on tyramine as one of the indicators of inappropriate food storage and processing conditions of milk products. In the current study, 25 dairy product samples were collected from local markets in Alexandria, Egypt, and were analyzed for bacterial contamination and prevalence of tyramine-producing bacteria by PCR using degenerate primers (DEC5/DEC3). While cream was the most contaminated dairy product, balady yogurt samples were the least contaminated ones. Different types of tyraminogenic bacteria have been isolated from the collected dairy products. Cream samples were contaminated with the widest variety of tyraminogenic bacteria among the isolated samples including Bacillus pumilis, Escherichia coli, Enterococcus faecium, and Proteus mirabilis. A total of 35 strains harboring tyrosine decarboxylase gene were detected with the identification of a novel tyramine-producing strain: Rummeliibacillus pycnus. These results indicate the promising application of degenerate primers (DEC5/DEC3) to detect tyramine production in dairy products, a goal that has been regarded as a challenge by manufacturers.

Dynamic monitoring of single-terminal norepinephrine transporter rate in the rodent cardiovascular system: A novel fluorescence imaging method.

Here, we validate the use of a novel fluorescent norepinephrine transporter (NET) substrate for dynamic measurements of transporter function in rodent cardiovascular tissue; this technique avoids the use of radiotracers and provides single-terminal resolution.Rodent (Wistar rats and C57BL/6 mice) hearts and mesenteric arteries (MA) were isolated, loaded with NET substrate Neurotransmitter Transporter Uptake Assay (NTUA) ex vivo and imaged with confocal microscopy.NTUA labelled noradrenergic nerve terminals in all four chambers of the heart and on the surface of MA. In all tissues, a temperature-dependent, stable linear increase in intra-terminal fluorescence upon NTUA exposure was observed; this was abolished by NET inhibitor desipramine (1 μM) and reversed by indirectly-acting sympathomimetic amine tyramine (10 μM). NET reuptake rates were similar across the mouse cardiac chambers. In both species, cardiac NET activity was significantly greater than in MA (by 62 ± 29% (mouse) and 21 ± 16% (rat)). We also show that mouse NET reuptake rate was twice as fast as that in the rat (for example, in the heart, by 94 ± 30%). Finally, NET reuptake rate in the mouse heart was attenuated with muscarinic agonist carbachol (10 μM) thus demonstrating the potential for parasympathetic regulation of norepinephrine clearance.Our data provide the first demonstration of monitoring intra-terminal NET function in rodent cardiovascular tissue. This straightforward method allows dynamic measurements of transporter rate in response to varying physiological conditions and drug treatments; this offers the potential to study new mechanisms of sympathetic dysfunction associated with cardiovascular disease.